Human prion diseases are unique in that they manifest as sporadic, iatrogenic and inherited neurodegenerative disorders. They express a variety of disease phenotypes characterized by the presence of an abnormal, protease-resistant isoform of the prion protein, PrPres. PrPres is encoded by the same PRNP gene as the normal, protease-sensitive prion protein, PrPc. The molecular basis for the conversion of PrPc to PrPres in human prion disease are largely unknown. The discovery of multiple PRNP mutations and the initial findings of distinct PrPres subtypes in different disease phenotypes suggest that changes in the PrP structure may underlie the phenotypic diversity in prion diseases. Our long-term objective is to define the exact structural features that are critical to the formation of PrPres in the disease state, with implications for rational design of effective treatment for these fatal disorders. The naturally occurring and disease-causing PRNP mutations have provided us with a rara opportunity to probe the relationship between the structural abnormalities of PrPres and the disease phenotypes they cause. The present research project proposes to examine this issue in four specific aims. Specific Aim 1 deals with the characterization of the major PrPres subtypes with respect to the differences in protease cleavage sites and mutation-specific chemical modifications. PrPres will be purified from brains of subjects with familial prion diseases. The primary structure and potential covalent modifications of various PrPres subtypes will be examined by multiple approaches, including enzymatic digestion, high performance liquid chromatography, N-terminal protein sequencing and mass spectrometry. In Specific Aim 2 the secondary structure of PrPres with different mutations will be defined. Spectroscopic methods such as Fourier transform infrared and circular dichroism will be used to characterize and compare the conformational properties of PrPres subtypes. Specific Aim 3 examines the conversion of PrPc to PrP res and the mechanism by which mutations affect the conversion process. A cell-free conversion system will be used to study the formation of nascent PrPres by in vitro incubation of radiolabeled, recombinant PrPc with the pre-existing, purified PrPres. Specific Aim 4 focuses on the characterization of the truncated and potentially amyloidogenic PrP derivatives that result from the cellular processing of the mutant PrP. Human neuroblastoma cells expressing the mutant PrP will be used to identify truncated forms of PrP by SDA/PAGE and immunoblot analysis. The identified PrP derivatives will be purified an characterized by N-terminal sequencing and mass spectrometry. These studies will lead to a better understanding of the molecular basis for phenotypic diversity in human prion diseases.